The present invention relates generally to flow cytometers and, in particular, relates to a method and apparatus for producing a consistent droplet break-off point.
Flow cytometers are used to analyze particles, such as blood cells, in a liquid suspension. For example a particular blood cell of interest may be present among various types of blood cells. In order to physically separate cells of interest, the cell suspension is presented within a flow cytometer""s flow cell through a sample injection tube where it is hydrodynamically focused within a pressurized sheath stream of electrolyte solution. This action produces a laminar flow of cells which is subjected to illumination at an analysis location, such as by a laser beam. To enhance the distinctive light reflective characteristics of the various cell types, the cells may be treated with fluorescent dyes or markers prior to testing with the flow cytometer. The flow cytometer measures the amount of light scattered in the forward and in the 90 degree angle directions from the laser and from any other emitted light that would result from laser excited fluorochromes which may be present on or in the cell of interest. Additionally, the instrument measures the amount of fluorescent light emitted by each particle as well as the corresponding light scatter patterns. These signals are collected in the form of light energy, and converted to electrical energy, and subsequently digitized and plotted on user-defined histograms.
Flow cytometric cell sorters thus have the ability to separate or identify particles of interest from other unwanted particles. In particular, the cell sorter is equipped with a bimorph crystal and an electric oscillator that vibrate the sample stream, causing it to break into free droplets at a specific user-defined droplet break-off point after having been identified for subsequent separation or sorting. The flow cytometer operator determines the population to be sorted during analysis by setting a user-defined sort region identifying particles that meet specific light-scatter and fluorescence criteria. The instrument is also programmed to apply an electrical charge to the droplet containing those particles of interest at a point downstream of the analysis location. As the now charged droplet, containing the particle of interest, then moves down into the area of the cell sorter""s oppositely-charged deflection plates. It is pulled away from the uncharged droplets containing unwanted cells, and pulled towards the oppositely charged deflection plate, and onto a glass microscope slide or into a sample collection tube. The uncharged droplets containing unwanted particles flow into a drain and into a waste collection tank.
One example of such a flow cytometer is described in U.S. Pat. No. 5,367,474, entitled xe2x80x9cFlow Cytometer,xe2x80x9d and assigned to Coulter Corp, Miami, Fla., the disclosure of which is hereby incorporated by reference as if set forth in its entirety herein.
During a routine period of instrument preparedness (usually an 8 hour day for a core facility) the sort stream of conventional cytometers becomes unstable. At the start-up of the sorter instrument, the crystal""s oscillation frequency will be established within a reasonable time. Sort purity and recovery are at acceptable levels within the time that the bimorph crystal, also known as a crystal assembly or piezoelectric crystal, is stable and producing a stable sort stream. As used herein, a stable sort stream is one whose droplet break-off point, defined as that location at which sheath and enclosed sample stream first start to produce separate and distinct droplets, is consistent, thereby facilitating a reliable separation of particles of interest. However, after a period of time, this droplet break-off point will change and cannot be recovered, thereby facilitating unreliable results.
Once this occurs, no current method exists for returning the sort stream""s stability to an operational state short of continually changing the bimorph crystal""s rate of oscillation. Doing so, however, requires a video camera with high resolution to examine minute changes in the droplet break-off point that are sufficiently miniscule so as to not adversely affect the reliability of the separation, while providing an indication that the oscillation frequency is fluctuating. Software logic is also necessary to vary the supply current to the crystal, along with a monitor is to examine the location of the droplet break-off point. This is undesirable in that the resources required are expensive, and is reactive to changes in oscillation frequency rather than being proactive.
What is therefore needed is a stable cytometer that proactively produces a consistent droplet break-off point to enable the reliable determination of a particle of interest.
The present invention recognizes that significant temperature fluctuations in the ambient environment surrounding the piezoelectric crystal occur due to significant changes in ambient room temperature and instrument temperature changes caused by heat-producing components within the instrument. This results in corresponding temperature fluctuations of the crystal itself. When these temperature fluctuations are too large, the crystal and the sort stream become unstable and the results unreliable. Depending on the quality of the crystal, temperature fluctuations as little as 1xc2x0 C. may affect the reliability. Accordingly, the preferred embodiment of the present invention comprises a cytometer that maintains the temperature of the piezoelectric crystal within a suitable range so as to maintain the stability of the resultant sort stream produced by the cytometer, thereby ensuring accurate results.
In accordance with one aspect of the invention, a flow cytometer includes a flow cell body having an inlet end for receiving sample particles, and an outlet end for delivering a stream of the sample particles to a analysis location, a laser mechanism directing a laser beam towards the sample particles at the analysis location, a sensor operable to measure the optical characteristics of the sample particles to identify select ones of the particles of interest, an actuator in mechanical communication with the flow cell body and oscillating at a frequency sufficient so as to separate the particles at a predetermined separation location, a temperature regulator in thermal communication with the actuator and operable to maintain the actuator within a predetermined temperature range.
This and other aspects of the invention are not intended to define the scope of the invention for which purpose claims are provided. In the following description, reference is made to the accompanying drawings, which form a part hereof, and in which there is shown by way of illustration, and not limitation, a preferred embodiment of the invention. Such embodiment also does not define the scope of the invention and reference must be made therefore to the claims for this purpose.